1. Field of the Invention
The present invention relates to novel substrates for determination of enzyme activity which are useful for assaying enzyme activity of trypsin, .alpha..sub.2 -macroglobulin-trypsin complex (hereinafter simply referred to as .alpha..sub.2 M-Try), or the like. The present invention also relates to intermediates for synthesis of the substrates and a process for producing the intermediates.
2.Related Art Statement
A method for determining enzyme activities of trypsin, urokinase, thrombin, etc. in human plasma involves assay using a substrate that releases a color-forming compound by the action of enzyme. According to this method, enzyme is reacted with a substrate to release a color-forming compound. An absorbance at a given wavelength that the color-forming compound absorbs is measured to determine an activity of enzyme as the analyte.
It is required that a substrate for assaying enzyme activity used in such a method should have good solubility in water or buffer, in addition to good reactivity, high selectivity, high sensitivity, high specificity to enzyme and easy detectability of its degradation products.
Many substrates have been developed heretofore as those for determination of enzyme activity. For example, proteins such as gelatin and hemoglobin, etc. have been used in the past as substrates for the detrmination of trysin. Howver, it is inappropriate to assay for trypsin activity in the pancreatic juice or duodenal juice using these substrates, since other proteinases, for example, chymotrypsin, elastase, etc., are present in such a juice.
It was reported by Bergman et al. that trypsin has the action of amidase and esterase, in addition to its proteolytic activity [J. Biol. Chem., 130, 81-86 (1939)]. Since then, many synthetic substrates (e.g., Benzyl-Arg-NH2, p-Toluenesulfonyl-Arg-OMe, Benzyl-D, L-Arg-p-Nitroanilide, p-Toluenesulfonyl-Arg-p-Nitroanilide, etc.) have been developed. However, many of these substrates cause cross reaction with serine proteases present in samples such as serum or ascitic fluid or with substances exhibiting similar enzyme activity, for example, thrombin, factor Xa, complement, kallikrein, etc. In addition, the desired reactivity with trypsin itself is insufficient so that a prolonged period of time is required for the measurement and reproducibility is questionable. Thus, these substrates cannot withstand practical use.
In recent years, arginylanilide derivatives such as Benzyloxycarbonyl-Val-Gly-Arg-p-Nitroanilide (CHR-TRY, Pentapharm Corp., U.S. Pat. No. 4,278,762), Benzyl-Ile-Glu (.gamma.--OH and --OCH.sub.3)-Gly-Arg-p-Nitroanilide.HCl (S-2222, Kabi Inc., J. Gastroent., 5, 533 (1970), Bergstrom, K), etc. have been developed as substrates for assaying trypsin activity. However, these derivatives encounter problems in selectivity, reactivity, solubility, etc. Moreover, the arginylanilide derivatives are expensive and not satisfactory yet.
In Japanese Patent Application KOKAI (Laid-Open) No. 59-106446, there are proposed color-forming substrates represented by formula: ##STR2## wherein X represents H or a protective group conventionally used for peptide synthesis; and A and B represent an amino acid or a residue of its derivative, as substrates for determination of enzyme activity of thrombin, kallikrein, urokinase, plasmin or the like. Substrates of ester type wherein R represents --OC.sub.n H.sub.2n+1, those of amide type wherein R represents --NHC.sub.n H.sub.2n+1 and those of amide type wherein R represents an amino acid residue are described therein; however, carboxyl type substrates wherein R represents OH are not found.
It is considered that carboxyl type substrates for determination of enzyme activity would be preferable, because of their solubility in water or buffer. It is also considered that substrates in the form of carboxyl type would exert high specificity to enzyme. Therefore, it has been desired to develop carboxyl type substrates.